All living creatures, including human beings, are made of cells. A majority of life forms exist as single cells that perform all functions necessary to continue independent life. Most cells are far too small to be seen with the naked eye and require the use of a high-power microscope to observe their behavior. Since their invention in the late 1500s, light microscopes have enhanced our knowledge of biology, human physiology and anatomy, biomedical research, medical diagnostics, and materials science. The science of microscopy has advanced to include a variety of techniques to enhance resolution.
Continuing advances in microbiology require a closer and closer study of biochemical events that occur on a cellular and intracellular level. The challenge in microscopy today is not only the enhancement of finer and finer resolution, but also the development of techniques for observing biochemical events in real time, as they happen, without destroying the biological specimen in the process.
Resolution is the ability of a microscope to distinguish between two objects that are very close together. A microscope with a resolution of 1,000 Å (1,000 Angstroms; equal to 100 nanometers or 100×10−9 meters), for example, can make objects as close together as 100 nanometers independently visible. Objects and features smaller than 100 nanometers cannot be resolved (i.e., distinguished). The typical resolution or practical resolving power of several types of microscopes currently available are approximately 2,000 Å for visible light microscopes, 1,000 Å for ultraviolet microscopes, 150 Å to 300 Å for scanning electron microscopes, and 2 Å to 4 Å for transmission electron microscopes.
The ultraviolet microscope offers finer resolution and better magnification than an ordinary light microscope, but it has serious disadvantages for the study of living specimens. Ultraviolet light damages or kills many kinds of living biological specimens, making observation of many biological processes impossible. When ultraviolet light strikes a specimen, it excites fluorescence within the molecules of the specimen so that the specimen itself emits a fluorescent light. If the specimen does not produce fluorescence naturally, then it must be stained with a fluorescent dye. Many fluorescent dyes bind strongly to elements such as enzymes within living cells, changing their qualities and significantly altering the cellular biochemistry. Other dyes produce too much fluorescence or absorb too much of the ultraviolet light to be useful.
The operation of an ultraviolet microscope requires a great deal of skill. Because ultraviolet light damages the human eye, the image can only be observed by ultraviolet video cameras or specially-equipped still cameras. Also, the quartz optics required for ultraviolet microscopes are typically much more expensive than the glass components used in visible light microscopes.
Because most bacteria and viruses are too small to be seen even with an optical microscope, an electron microscope is generally used to view such organisms. Although electron microscopes offer very fine resolution, the specimen typically must be prepared by high-vacuum dehydration, freezing, impregnation with heavy metals, and is subjected to intense heat by the electron beam, making the observation of living specimens impossible. The dehydration process also alters the specimen, leaving artifacts and cell damage that were not present in nature. Also, in order to view the steps in a biological process, dozens of specimens must be viewed at various stages in order to capture each desired step in the process. Each selected specimen must then be prepared, using a process that can take up to two hours per specimen.
The high cost of an electron microscope represents another barrier to its use in the life sciences. Electron microscopes are large and often occupy an entire room. The operation and adjustment of an electron microscope, like an ultraviolet microscope, requires highly-skilled technicians, introducing yet another cost of maintaining and staffing an electron microscopy facility. Thus, the electron and ultraviolet microscopes available today generally do not offer a technique for observing living, unaltered biological specimens in real time.
Many biological properties can only be viewed in living cells. Such properties include transport, streaming, Brownian motion, diffusion, phagocytosis, pinocytosis, mitosis, immuno-fluorescence, and cell interactions. Biomedical technologies including, but not limited to, gene therapy, artificial insemination, new drug development, cell culturing and cloning, cell regeneration, implantation, biodetecting, and biotherapeutics require the visualization of living cells and cellular processes. While the nature of these phenomena can sometimes be inferred through examining electron micrographs before and after these processes occur, such processes are more preferably studied in depth while they are occurring.
Fluorescent microscopes can be useful to the study of bacteria, animal, and plant cells, as they show primary fluorescence (autofluorescence) when illuminated with ultraviolet light. A fluorescent microscope is a microscope for observation of small objects by a light of their fluorescence. Fluorescence is a short time luminescence with a lifetime of about 10−8-10−9 seconds, in contrast with phosphorescence that has a much longer lifetime. Fluorescence is most commonly generated by excitation with light. The emitted fluorescence light normally has a longer wavelength than that of the exciting light. Three important steps can divide the process of fluorescence. First, a molecule is excited by an incoming photon during the first few femtoseconds (10−15 seconds). During the next few picoseconds (10−12 seconds), the molecule goes through a vibrational relaxation of an excited state electron to the lowest energy level of the intermediate states. Finally, emission of a longer wavelength photon and recovery of the molecule into the ground state occurs during a few nanoseconds (10−9 seconds). The whole process from excitation of the molecule by an excitation light (EL) to emission of a longer wavelength fluorescent light (FL) is used for fluorescent microscopy.
Initial studies on fluorescent microscopy were carried out during the early part of the twentieth century by August Kohler and Carl Reichert. The first practical fluorescent microscopes were demonstrated by Otto Heimstadt and Heinrich Lehmann in 1911. A short time later, Stanislav von Provazek and Alfred Coles used organic dyes termed “fluorochromes” for securing secondary fluorescence. The secondary fluorescence for the study of sections of tissues and organs stained with fluorochromes was thoroughly investigated by Max Haintinger. However, in 1941 the real revolution in fluorescent microscopy occurred when Albert Coons developed a technique for labeling antibodies with fluorescent dyes (“fluorescent labeled antibodies”), and thus introduced the field of immunofluorescence, which is now a standard method.
The main function of a fluorescent microscope is to illuminate a sample with light of a specific wavelength (excitation light), excite the molecules of the sample with a fluorescent light, and then separate a weak emitted fluorescence from the excitation light, so that the emitted fluorescence can be observed. A special light source and the presence of two filters typically characterize the optical pathways of the fluorescent microscope: one filter is placed before a condenser and the other filter is placed after the objective. The first filter transmits only exciting radiation, and the second filter transmits only emitted fluorescent light. Thus, the excitation light incident on a sample is removed, while fluorescent light is directed to the observer's eye, or to a recording device. The light source should provide a short-wavelength light such as UV and/or blueviolet light. Currently, there are two different optical designs of fluorescent microscopes in common usage: one uses a transmitted light illumination (“dia-fluorescence microscopy”) and the other employs a reflected light (“epi-fluorescence microscopy”).
The light of the wavelengths required for fluorescence excitation are selected by an excitation filter, which transmits only exciting light and suppresses light of all other wavelengths. A certain part of the exciting light is adsorbed by the sample and almost instantaneously reemitted at longer wavelengths as fluorescence light. A barrier filter transmits the fluorescence light (emission light). The rest of the excitation light which passes through or reflects from the sample is absorbed by the barrier filter. As a result, a color image of the sample is observed (or recorded) against a dark background.
Early fluorescence microscopes were generally brightfield transmitted light microscopes equipped with excitation and barrier filters. The transmitted light fluorescence microscope was greatly improved by using a darkfield condenser. A darkfield condenser projects light onto the sample at oblique angles, which prevents excitation light from directly entering the objective. Certain difficulties of the conventional transmitted light fluorescence light microscope made the reflected light fluorescence microscope the instrument of choice by many users. Although transmitted light fluorescence microscopy has proven valuable in various applications, the technique has some disadvantages, which include the following: (1) the numerical aperture of the objective needs to be reduced in order to prevent excitation light from entering the objective, which in turn reduces light intensity and resolution; (2) the conventional darkfield method is very wasteful of light (i.e., not very efficient); (3) some users find it difficult to align a darkfield condenser; (4) the emitted fluorescent light passes through the sample before reaching the objective, and therefore the light is partly absorbed and scattered, which results in diffuse and less intense images; and (5) the conventional darkfield technique precludes the use of simultaneous fluorescence viewing together with phase microscopy or Nomarski differential interference contrast microscopy. Because of all of these problems of using darkfield transmitted fluorescence microscopy (dia-fluorescence microscopy), brightfield reflected fluorescence microscopy (epi-fluorescence microscopy) is generally preferred.
The Nature of Light
Light is sometimes referred to as a type of electromagnetic radiation because a light wave consists of energy in the form of both electric and magnetic fields. In addition to the light we can see, the electromagnetic spectrum includes radio waves, microwaves, and infrared light at frequencies lower than visible light. At the upper end of the spectrum, ultraviolet radiation, x-rays, and gamma rays travel at frequencies higher than visible light.
Wavelength is the distance between any two corresponding points on successive light waves. Wavelength is measured in units of distance, usually billionths of a meter. The human eye can see wavelengths between 400 and 700 billionths of a meter. Frequency is the number of waves that pass a point in space during any time interval, usually one second. Frequency is measured in units of waves per second, or Hertz (Hz). The frequency of visible light is referred to as color. For example, light traveling at 430 trillion Hz is seen as the color red.
The wavelength of light is related to the frequency by the simple equationf=c/L where c is the speed of light in a vacuum (299,792,458 meters per second), f is the frequency in Hertz (Hz) or cycles per second, and L is the wavelength in meters.Microscope Resolution
The resolution or resolving power of a light microscope can be calculated using Abbe's Formula:D=L/2NA where D is the resolving power of a microscope in meters, L is the wavelength in meters of the incident light, and NA is the numerical aperture of the microscope. The numerical aperture, generally, indicates the angle at which light strikes the specimen being viewed.Light Scattering
When a light wave passes through a specimen, most of the light continues in its original direction, but a small fraction of the light is scattered in other directions. The light used to illuminate the specimen is called the incident light. The scattering of incident light through various specimens was studied by Lord John William Strutt, the third Baron Rayleigh (Lord Rayleigh) in the late 1800s, and later by Albert Einstein and others.
Lord Rayleigh observed that a fraction of the scattered light emerges at the same wavelength as the incident light. Because of this observation, light that is scattered at the same wavelength as the incident light is a phenomenon called Rayleigh scattering (also called resonant scattering or elastic light scattering).
In 1922, Arthur H. Compton observed that some of the scattered light has a different wavelength from the incident light. Compton discovered that, when light passes through a specimen, some of the light scatters off the electrons of the specimen molecules, producing scattered light in the X-ray region of the spectrum.
Raman Scattering
In 1928, Professor Chandrasekhara V. Raman and Professor K. S. Krislman discovered that the scattered light observed by Compton was caused by vibrations within the molecules of the specimen. Because of his discovery, light that is scattered due to vibrations within the molecules of a specimen is a phenomenon called Raman scattering (also called non-resonant or inelastic light scattering). In 1930, Raman received the Nobel Prize in Physics for his discovery.
When a specimen is bombarded with incident light, energy is exchanged between the light and the molecules of the specimen. The molecules vibrate, producing the phenomenon known as Raman scattering. The molecular vibrations cause the specimen itself to emit scattered light, some of which scatters at a higher frequency (f+Δf) than the incident light frequency (f), and some of which scatters at a lower frequency (f−Δf). The Δf represents the change in frequency (sometimes called the frequency shift) produced by Raman scattering.
In summary, when incident light strikes a specimen, the scattered light includes Rayleigh-scattered light at the same frequency (f) as the incident light, higher frequency (f+Δf) Raman-scattered light, and lower-frequency (f−Δf) Raman-scattered light.
Intensity Depends on the Specimen
Because Raman-scattered light is produced by molecular vibrations within the specimen, the intensity of the Raman-scattered light varies depending upon the type of specimen being viewed. For example, a specimen of blood cells may produce high-intensity Raman-scattered light, while a specimen of skin cells may produce lower-intensity Raman-scattered light. One way to harness the resolving power of Raman-scattered light is through the use of a darkfield condenser to focus the incident light on the specimen.
Darkfield Microscopy
Darkfield observation in microscopy uses a condenser to shape the incoming light into a cone of light with its vertex or focal point directed toward the specimen. A darkfield condenser usually includes a centrally-disposed opaque stop and one or more internal lenses or mirrors to shape the light into the desired hollow cone shape. The opaque stop blocks a large portion of the incoming light, allowing only a hollow cylinder of light enter the condenser.
In darkfield microscopy, if there is no specimen on the microscope stage and the numerical aperture of the condenser is greater than that of the objective, the cone-shaped light rays converge at or near the stage, then diverge beyond the stage such that they do not enter the objective lens, and the field of view will appear dark. When a specimen is present, the cone-shaped light rays strike the specimen and are scattered, diffracted, reflected, and/or refracted by the various features of the specimen that create optical discontinuities. Some of these light rays enter the objective lens, revealing the features of the specimen, which appear against a dark background.
Many types of condensers are available and in use today. In a cardioid darkfield condenser, the incoming light passes around a central opaque stop, strikes a convex mirror, and then strikes an internal concave mirror having a spherical surface and a cardioidal surface. A paraboloid darkfield condenser works much like the cardioid, except the internal mirror is parabolic in shape. In an Abbe darkfield condenser, the incoming light passes around a central opaque stop, then passes through a generally convex lens, and finally passes through a second lens. The Abbe darkfield condenser may include a variable internal aperture. Other types of darkfield condensers include the bicentric, bispheric, Cassegrain, spot-ring bicentric, and Nelson-Cassegrain.
After the incoming light passes around the central opaque stop, the light is shaped like a thin-walled hollow cylinder. The hollow cylinder of light then strikes the internal lenses or mirrors, where the light is refracted into the desired hollow cone of light. The refraction of light usually takes place near the perimeter of the internal lens elements, where optical correction is often the poorest. Therefore, to obtain a precise hollow cone of light, the internal lenses are made with great precision to avoid creating anomalies. Precision grinding of lenses and mirrors greatly increases the cost of a darkfield condenser.
The opaque stop inside a darkfield condenser is carefully aligned in the center, to create a uniform hollow cylinder of light. A poorly-centered stop can skew the hollow cone of light, causing uneven illumination and other undesired optical effects that interfere with the image quality. Because the condenser requires extremely precise alignment, it often takes a highly-skilled operator to align a darkfield microscope system. The alignment sensitivity also makes the darkfield system vulnerable to minute vibrations.
Because the opaque stop blocks a large portion of the incoming light, a powerful light source is usually required. In addition to being wasteful, high-power light sources are expensive to operate and maintain, and the excess heat generated may cause undesirable heating of the condenser body, the microscope stage, and the specimen.
Thus, it can be seen that needs exist for improved devices, systems, and methods for viewing living biological specimens with better resolution, including their cellular structures and functions in real time. It is to the provision of such devices, systems, and methods meeting these and other needs that the present invention is primarily directed.